Naturally occurring interferons (IFNs) are species-specific proteins, often glycoproteins, produced by various cells upon induction with viruses, double stranded RNAs, other polynucleotides, antigens and mitogens. Interferons exhibit multiple biological activities such as antiviral, antiproliferative, immunomodulatory and anticellular functions. At least three distinct types of human interferons have been indentified and characterized in terms of their anti-viral, anti-growth and activation of natural killer cell (NK) activities. They are produced by leukocytes, lymphocytes, fibroblasts and the immune system and are classified as .alpha., .beta. and .gamma. interferons. These are reported to be different proteins coded for by distinct structural genes.
Native human .beta.-interferon is generally produced by superinducing human fibroblast cultures with poly-IC (polyriboinosinic acid and polyribocytidylic acid) and isolating and purifying native human .beta.-interferon thus produced by chromatographic and electrophoretic techniques. Proteins or polypeptides which exhibit native .beta.-interferon-like properties may also be produced using recombinant DNA technology by extracting poly-A-rich 12S messenger RNA from virally induced human cells, synthesizing double-stranded c-DNA using the m-RNA as a template, introducing the c-DNA into an appropriate cloning vector, transforming suitable microorganisms with the vector, harvesting the bacteria and extracting the IFN-.beta. therefrom. Nagola, S. et al., Nature, 284: 316 (1980); Goeddel, D. V. et al., Nature, 287: 411 (1980); Yelverton, E. et al., Nuc. Acid Res., 9: 731 (1981); Streuli, M. et al., Proc. Nat'l. Acad. Sci. (U.S. ), 78: 2848 (1981); European Pat. Application Nos. 28033, published May 6, 1981; 321134, published July 15, 1981; 34307 published Aug. 26, 1981; and Belgian Patent 837397, issued July 1, 1981 describe various currently used methods for the production of .beta.-interferon employing recombinant DNA techniques. The expressed proteins or polypeptides have been purified and tested and have been found to exhibit properties similar to those of native IFNs. Bacterially produced IFNs thus appear to have potential therapeutic use as antiviral and anti-tumor agents and the production of IFNs by such bacterial fermentations is expected to yield sufficiently large quantities of IFN at a relatively low cost of clinical testing.
Further, human IFN-.beta. genes have been altered by, for example, oligonucleotide-directed mutagenesis to produce IFN-.beta. protein analogs thereof, such as the human recombinant cysteine-depleted or cysteine-replaced interferon-.beta. analogs (muteins) disclosed in U.S. Pat. No. 4,588,585 issued May 13, 1986 to Mark et al. Specifically disclosed in that patent is the recombinant IFN-.beta. wherein the cysteine at position 17 is replaced by the neutral amino acid serine. That IFN-.beta. analog is IFN-.beta..sub.ser17.
Procedures for recovering and purifying bacterially produced IFNs are described in U.S. Pat. Nos. 4,450,103; 4,315,852; 4,343,735; and 4,343,736; and Derynck et al., Nature (1980) 287: 193-197 and Scandella and Kornberg, Biochemistry, 10: 4447 (1971). Generally with these methods the IFN is not produced in a sufficiently pure form and in sufficiently large quantities for clinical and therapeutic purposes and the resulting IFN preparations produced by recombinant DNA techniques have residual amounts of chemicals and considerable microheterogeneity.
Purification and activity assurance of precipitated heterologous proteins is also described by U.S. Pat. Nos. 4,511,502; 4,511,503; 4,512,922; and 4,518,526; and in European Patent 114,506.
Copending, commonly owned U.S. patent application Ser. No. 843,997 filed Mar. 25, 1986 entitled "Process for Recovering Refractile Bodies Containing Heterologous Proteins from Microbial Hosts" discloses a biochemical separation or recovery process in which refractile bodies containing microbially produced IFN-.beta. are separated or recovered from the microorganism hosts, and further discloses protocols for then purifying the isolated refractile bodies or refractile material.
Commonly owned U.S. Pat. No. 4,462,940, and copending commonly owned U.S. patent application Ser. Nos. 775,751 (filed Sept. 13, 1985), 923,425 (filed Oct. 27, 1986) and 923,432 (filed Oct. 27, 1986) further disclose processes for purifying and formulating recombinant IFN-.beta..
U.S. Pat. No. 4,343,735 to Menge et al. teaches a process for the purification of interferon by partitioning it in an aqueous multi-phase system in the presence of ion exchangers which are soluble in the system and are derivatives of polyethers.
U.S. Pat. No. 4,289,690 to Pestka et al. discloses processes for purifying proteins including native human leukocyte interferon by utilizing one or more high performance liquid chromatography steps employing as solvents alkanols, such as n-propanol. See also, Pestak et al., Pharmac. Ther., 29: 299-319 (1985); Langer et al., J. Investig. Dermatol., 83 (1): 1285-1365 (1984); and Pestka, S., Archives Biochem. Biophys., 221 (1): 1-37 (Feb. 15, 1983).
U.S. Pat. No. 4,289,689 to Friesen et al., discloses how to recover and purify human native .beta.-interferon by use of affinity chromatography and high pressure liquid chromatography.
In handling a biologically active protein, such as recombinant IFN-.beta., general considerations concerning the handling of proteins are relevant, including the necessity of preserving the protein's delicate tertiary structure in order to preserve biological activity, which requires the avoidance of denaturing pH conditions. E. coli expressed recombinant IFN-.beta. and analogs thereof are insoluble in solutions which are at a pH range of 6 to 9. Therefore, various processes and additives have been devised to solubilize these proteins.
In producing a recombinant protein such as IFN-.beta. which is to be administered therapeutically to humans or animals, considerations of purity and homogeneity of the final product are of the utmost concern. Reduction or elimination of minor IFN-.beta. species, and removal of both non-IFN-.beta. proteins and bacterial endotoxins, are of prime importance. Secondarily, in establishing or improving a purification scheme of a therapeutic, recombinant protein are considerations of efficiency and simplicity of the process.
The variations on the theme of protein purification have been explored for more than fifty years. The literature on this subject is extensive and a plethora of techniques is available to the practitioner, including ion exchange chromatography, adsorption chromatography, gel electrophoresis, ammonium sulfate precipitations, and gel filtration. Over the years there have been substantial improvements in the technology of conducting many of the foregoing methods, and in particular, it has been possible to automate and speed up the procedures related to column chromatography and development of electrophoresis gels. Despite these technical advances, and despite the large number of proteins which have been subjected to these procedures, the selection of a successful procedure, or more usually combination of procedures, for a particular protein found in a particular milieu has remained unpredictable, unselectable in advance, and subject to considerable experimentation in each particular case.
The instant invention incorporates a RP-HPLC method, specifically adapted for the purification of recombinant IFN-.beta., in purification processes essentially outlined in commonly owned, copending U.S. patent application Ser. Nos. 843,997 (filed Mar. 25, 1986) and 923,423 (filed Oct. 27, 1986), which applications are herein incorporated by reference. References to purification schemes for recombinant proteins also employing RP-HPLC are listed below.
U.S. Pat. No. 4,485,017 issued to Tan et al. discloses a process for the isolation and purification of native interferons wherein a partially purified preparation of native interferon is sequentially passed through an antibody affinity column and an RP-HPLC column.
U.S. Pat. No. 4,569,790 issued to Koths et al. discloses a process for recovering microbially produced IL-2 wherein the oxidized IL-2 is purified by RP-HPLC. A gradient solvent system comprising an organic acid such as acetic acid or trifluoroacetic acid (TFA) and an organic solvent such as propanol or acetonitrile is used to elute the IL-2 from the reverse-phase column.
Tarr et al., Anal. Biochem., 131: 99-107 (1983) describes the use of RP-HPLC resolution and recovery of cytochrome P-450 and bovine rhodopsin using ternary solvents, including acetonitrile and n-propanol and mixtures of the two.
Bennett et al., Biochem., 20: 4530-4538 (1981) describes the purification of two major forms of rat corticotropin (ACTH) to apparent homogeneity by RP-HPLC using solvent systems containing either TFA or heptafluorobutyric acid (HFBA) as hydrophobic counter-ions.
Bennett et al., Biochem., 197: 391-400 (1981) describes the isolation and analysis of human parathyrin wherein RP-HPLC employing solvent systems composed of aqueous acetonitrile containing TFA or HFBA as hydrophobic ion-pairing reagents is used.
Smith-Johannsen et al., J. IFN. Res., 3 (4): 473-477 (Nov. 4, 1983) discloses a procedure involving the chromatography of native human IFN-.beta. on an antibody affinity column followed by RP-HPLC. The RP-HPLC was performed wherein the elution solvent contained n-propanol in a linear gradient of between 0-100%, and wherein the C.sub.18 column was equilibrated with 100 mM formic acid. See also, Colby et al., J. Immunol., 133 (6): 3091-3095 (December, 1984) wherein recombinant IFN-.beta. is purified according to the Smith-Johannsen et al. method.
Heukeshoven et al., Chromatographia, 19: 95-100 (1985) discloses RP-HPLC for various virus proteins and other hydrophobic proteins wherein a proportion of 60% formic acid in all solvents was used and wherein 2-propanol or acetonitrile was the organic modifier for gradient elution.
Burgess et al., PNAS (USA), 79: 5753-5757 (October 1982) discloses the separation of two forms of murine epidermal growth factor by using RP-HPLC wherein the results of employing solvent systems containing either 0.2% trifluoroacetic acid or 0.2% heptafluorobutyric acid and 20 or 50% acetonitrile were compared.
Friesen et al., Arch. Biochem. Biophys., 206 (2):432-450 (February 1981) discloses the purification of native human IFN-.beta. by a combination of affinity chromatography and RP-HPLC wherein the reverse-phase column is equilibrated with pyridine-formic acid containing isopropanol and/or n-butanol.
Molnar (Ed.), Practical Aspects of Modern HPLC (Proceedings Dec. 7-8, 1981 in West Berlin): Freisen, H., "HPLC of Protein on Reverse Phase Exemplified with Human Interferons and Other Proteins: Review and Scope of a Method" (pp. 77-107) provides a review article on reverse phase chromatography of proteins, including interferons.
The instant invention provides improved purification processes incorporating RP-HPLC methods specific for recombinant IFN-.beta.. Said processes produce a highly pure IFN-.beta. product at very reduced levels of minor IFN-.beta. species, bacterial endotoxins and non-IFN-.beta. proteins. Said processes provide flexible options for simplifying and improving the efficiency of the purification of recombinant IFN-.beta., as well as increasing the purity and homogeneity of the product.